PROJECT SUMMARY/ABSTRACT Mitochondrial Complex II, also known as succinate dehydrogenase (SDH) is a membrane-bound heterotetramer (SDH-ABCD) whose four subunits are encoded by nuclear DNA. Complex II has a dual function in the cell by linking two essential energy-producing processes. As part of the TCA cycle the hydrophilic SDHAB domain oxidizes succinate to fumarate. This oxidation generates electrons that are transferred through the Fe-S clusters of the SDHB subunit to reduce ubiquinone within the SDHCD membrane domain to provide reducing equivalent to the electron transport chain needed for energy generation. As a key metabolic enzyme malfunction of Complex II is associated with debilitating neurodegenerative diseases and tumor formation. Although a significant amount of information is available on the structure and function of mature Complex II, there is gap in our understanding of how the enzyme assembles into a functional complex. In this project we will identify how various assembly factors work together to insert the flavin (FAD) and Fe-S redox centers into the SDHAB subunits and how this process is coordinated to assemble a functional Complex II. Aim 1: Our recent progress has provided insight into how assembly chaperones interact with the bacterial SdhA subunit to form the covalent FAD linkage needed for catalysis. We will expand on these findings to show how substrates enable protein domain movements to activate this assembly process and control catalysis in both bacterial SdhA and human SDHA subunits. This will be accomplished by x-ray crystallography, SAXS, and double electron electron resonance (DEER) spectroscopy of different intermediate assembly states. Aim 2: It is known that the Fe-S containing subunit requires specific assembly factors to incorporate its clusters. In this aim we investigate how the human SDHB (or bacterial SdhB) subunit incorporates its three unique Fe-S clusters localized into two separate protein domains. We also will determine whether the unique Complex II assembly chaperones assist with the insertion of the Fe-S clusters, or whether they stabilize the protein intermediates for interaction with the rest of the cellular Fe-S assembly machinery. Aim 3: Either malfunction of Complex II assembly or disassociation of the assembled complex during ischemia/reperfusion can contribute pathologies in mitochondrial function. In this aim we reveal mechanisms of how the SDHA and SDHB subunits form a functional subcomplex and aberrant activities associated with accumulation of the SDHAB subcomplex. A focus of this aim is how assembly factors and other proteins control the potentially deleterious reactive oxygen species that can be formed from the SDHAB subcomplex.